Microbial diversity in biofilm infections of the urinary tract with the use of sonication techniques

Authors


  • Editor: John Costerton

Correspondence: Veronika Holá, Microbiological Institute, Faculty of Medicine, Masaryk University and St Anne's University Hospital, Pekarska 53, CZ-65691 Brno, Czech Republic. Tel.: +420 54 318 3093; fax: +420 54 318 3089; e-mail: veronika.hola@fnusa.cz

Abstract

Infections of the urinary tract account for >40% of nosocomial infections; most of these are infections in catheterized patients. Bacterial colonization of the urinary tract and catheters causes not only the particular infection but also a number of complications, for example blockage of catheters with crystallic deposits of bacterial origin, generation of gravels and pyelonephritis. Infections of urinary catheters are only rarely single-species infections. The longer a patient is catheterized, the higher the diversity of biofilm microbial communities. The aims of this study were to investigate the microbial diversity on the catheters and to compare the ability to form biofilm among isolated microbial species. The next aim was to discriminate particular causative agents of infections of the urinary tract and their importance as biofilm formers in the microbial community on the urinary catheter. We examined catheters from 535 patients and isolated 1555 strains of microorganisms. Most of the catheters were infected by three or more microorganisms; only 12.5% showed monomicrobial infection. Among the microorganisms isolated from the urinary catheters, there were significant differences in biofilm-forming ability, and we therefore conclude that some microbial species have greater potential to cause a biofilm-based infection, whereas others can be only passive members of the biofilm community.

The number of infections of urinary catheters (IUC) has increased during the last decade. Urinary catheters have become the second most frequently used foreign body inserted into the human body. Over 40% of nosocomial infections are infections of the urinary tract, especially infections in catheterized patients (Gorman & Jones, 1991). Despite good aseptic management, c. 50% of patients have bacteriuria during the first 10–14 days of catheterization (Morris & Stickler, 1998).

The risk of urinary tract infections is significantly higher in long-term inserted catheters. The bacteria can more easily attack the urinary tract and the urinary bladder in the presence of a catheter (Tunney et al., 1999). Also, other complications can accompany bacterial colonization of catheters and the urinary tract, for example blockage of catheters with crystallic deposits of bacterial origin, generation of gravels and pyelonephritis (Gorman & Tunney, 1997).

Adhesion of bacteria to the catheter depends on many factors, including surface charge, hydrophobicity or hydrophilicity of the catheter and the bacterial cell, specific genes for adhesion, etc. (Liedl, 2001). Proteins and other components of human urine, which can physicochemically bind to the surface of the catheter and facilitate receptor-dependent binding of bacteria, also play an important role. The uroepithelium is easily colonized by bacteria (Gorman & Jones, 1991), especially if the epithelium is damaged by the catheter or by crystallic deposits.

The risk of infection is dependent on the length of catheterization and also on catheter management (Tenke et al., 2006). The longer the patient is catheterized, the higher the diversity shown by the biofilm microbial community. Patients with long-term catheterization are commonly infected by complex communities of bacteria (with a dominance of gram-negative rods). Catheter infections of the urinary tract are caused most commonly by faecal microbial communities – gram-negative rods (Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Pseudomonas aeruginosa, Proteus mirabilis, etc.) and enterococci (especially Enterococcus faecalis) (Tenke et al., 2006). Less often, the urinary infections result in colonization by other species of bacteria, for example Staphylococcus epidermidis and Streptococcus agalactiae and yeasts (Candida albicans).

Increased pathogenicity of these microorganisms is caused by the presence of many virulence factors, particularly the ability to form biofilm, the ability to coaggregate, or the ability to withstand the effect of antibiotics. Strains producing extended-spectrum-β-lactamases (ESBL, ampC) and MRSA can be a particularly important problem.

The diagnosis of IUC is based on culture proof of microbial agents from catheterized urine (including quantification) and on the examination of urinary catheters. For IUC diagnosis, the preculture of the catheter in liquid medium and subsequent inoculation of precultured liquid medium to solid media are commonly used. One disadvantage of this method is that one fast-growing microorganism can overgrow other species in the sample, and thus this method can lead to misinterpretation of the infection as a single- or a dual-species infection. The second disadvantage is the impossibility of quantification of microorganisms in the sample. A solution may be the use of techniques based on sonication of the catheter.

The aims of this study were to investigate microbial diversity on urinary catheters using sonication techniques compared with the commonly used method of precultivation of the catheter in a liquid medium. The next aim was to compare biofilm-forming ability among isolated microbial species, and in this way, to differentiate particular causative agents of infections of the urinary tract and their importance as biofilm formers in the microbial community on the urinary catheter.

Materials and methods

During the years 2007–2009, we collected strains isolated from the urinary catheters of patients of St Anne's University Hospital. We collected 535 culture-positive catheters. The catheters were processed simultaneously in two ways – using the common precultivation technique and the sonication technique.

Precultivation technique

The catheter was precultured in brain–heart infusion (BHI, Oxoid, UK) overnight. The liquid medium was subsequently inoculated to solid media to isolate individual strains. All isolated strains were identified to the species/genus level using conventional biochemical tests (Micro-LA-tests, Lachema, CZ; API Biomerieux, FR).

Sonication technique

We used the sonication protocol based on the procedure described previously by Sherertz et al. (1990). We sonicated the catheter in 5 mL of BHI for 5 min, and then vortexed for 2 min and sonicated for another 5 min. The suspension was subsequently inoculated to solid media to isolate individual strains. All isolated strains were identified to the species/genus level using conventional biochemical tests (Micro-LA-tests, Lachema, CZ; API Biomerieux, FR).

Biofilm examination

Culture conditions corresponded to those described in Stepanovićet al. (2007); briefly, all strains were cultivated in microtiter tissue culture plates at a temperature of 37 °C for 24 h in BHI with 4% of glucose. Each strain was cultivated in triplicate. After cultivation, the wells of the microtiter plates were washed three times and the biofilm layer was fixed by air-drying. The fixed biofilm layer was stained with crystal violet and the biofilm positivity was assessed quantitatively by OD595 nm assessment. On the basis of the OD of the negative control, the cutoff value (ODc) was calculated – OD of negative control plus 3 × SD. On the basis of ODc, the strains were divided into four groups – strains without biofilm formation (OD<ODc), strains with weak biofilm formation (ODc<OD<2ODc), strains with intermediate biofilm formation (2ODc<OD<4ODc) and strains with strong biofilm formation (4ODc<OD).

Results and discussion

We collected 535 culture-positive catheters and isolated 1555 microbial strains using the sonication technique. The number of isolated strains per catheter ranged between one and six. One strain was isolated from 69 catheters, two strains were isolated from 141 catheters, three strains were isolated from 160 catheters, four strains were isolated from 108 catheters, five strains were isolated from 50 catheters and six strains were isolated from seven catheters. We isolated 39 different microbial taxa; the spectrum of isolated microorganisms is listed in Table 1. The most frequently isolated were E. faecalis (294 strains), E. coli (213 strains), P. aeruginosa (148 strains) and Candida albicans (141 strains).

Table 1.   Isolated microbial species and their biofilm formation.
 Sum of strainsBF 0 (%)BF 1(%)BF 2 (%)BF 3 (%)
  • BF 0, no biofilm producer, OD<0.172; BF 1, weak biofilm producer, OD=0.172–0.344; BF 2, intermediate biofilm producer, OD=0.344–0.688; BF 3, strong biofilm producer, OD>0.688.

  • *

    In species with number of isolates lower than 10, only absolute numbers are shown.

Acinetobacter baumanii*80206
Bordetella bronchiseptica*10010
Burkholderia cepacia*20002
Candida albicans1413.917.121.157.9
Candida glabrata3810.55.313.370.9
Candida krusei*60060
Candida parapsilosis*50005
Candida tropicalis339.20090.8
Citrobacter diversus*10100
Coagulase-negative staphylococci2021.04.014.980.2
Enterococcus faecium933.116.220.460.3
Enterococcus faecalis2943.802.194.2
Enterobacter aerogenes16031.225.043.8
Enterobacter cloacae20010.015.075.0
Enterobacter dissolvens*20011
Enterobacter kobei*10001
Enterobacter sp.*60213
Escherichia coli2131.921.142.334.7
Hafnia alvei*10001
Klebsiella ornithinolytica*10100
Klebsiella oxytoca*81214
Klebsiella planticola*42002
Klebsiella pneumoniae972.012.439.246.4
Klebsiella sp.808.86.232.552.6
Kluyvera cryocrescens*10010
Morganella morganii1816.0028.255.9
Pantoea agglomerans*70115
Proteus mirabilis673.003.094.0
Proteus vulgaris*60024
Providentia stuabii*10010
Providentia rettgeri*10001
Pseudomonas aeruginosa1492.09.414.174.5
Ralstonia picketii*11000
Raoutella terrigena*41201
Serratia marcescens*10001
Staphylococcus aureus15000100.0
Stenotrophomonas maltophilia60213
Viridans Streptococcus sp.*40013
Yersinia rohdei*11000

Using the precultivation technique for examination of the same catheters, we isolated 727 microbial strains. The number of isolated strains per catheter ranged between one and four. One strain was isolated from 355 catheters, two strains were isolated from 170 catheters, three strains were isolated from eight catheters and four strains were isolated from two catheters. We isolated 27 different taxa (data not shown). The most frequently isolated were P. aeruginosa (102 strains), E. faecalis (41 strains) and E. coli (37 strains).

These results show that the sonication technique is more reliable for the examination of biofilm infections. The higher number of isolated microbial species from one catheter can be caused by the biofilm mode of growth as well as by the lower sensitivity of the precultivation techniques in terms of the microbial spectrum. The bacteria from the biofilm layer are not so easily cultivated and thus the sonication and so disruption of the biofilm layer can lead to better results. Overgrowing of some fast-growing microorganisms during the precultivation phase can suppress the growth of other species and thus lead to the lower sensitivity of the precultivation techniques. For both of these reasons, the infection can be misinterpreted as a single- or a dual-species infection only.

The ability to form biofilm was present in most isolates. More than 68% of all isolates were strong biofilm producers and 19% were intermediate biofilm producers. Only 3.6% of the strains isolated from IUC were not able to form biofilms. Biofilm formation differed among particular microbial species. Several species showed a high ratio of biofilm-positive strains, whereas others showed a lower ratio. The differences in biofilm formation among microbial species were statistically significant (anova, P=0.0031). The highest ratios of strong biofilm-positive strains were found in the species E. faecalis (94.2%), P. mirabilis (94%), Candida tropicalis (90.8%) and Staphylococcus aureus (100%). Escherichia coli showed a low ratio of strong biofilm-positive strains (34.7%).

The strongly biofilm-forming strains appear to be responsible for biofilm production in mixed-species biofilms. These species seem to be primary colonizers and coaggregate with other species, or alternatively, just provide shelter to other species that are only weak biofilm producers, thereby building up the mixed-species biofilm community. Our experiences (Ruzicka et al., 2004) as well as those of other researchers (Frebourg et al., 2000) indicate high levels of correlation between the ability of the microorganism to form biofilm and the clinical relevance of the strain; therefore, the differentiation of risk groups of bacteria in complex mixed-species biofilms of urinary catheters enables us to focus the therapy of these infections in the correct way. The sonication culture techniques are an appropriate method for the examination of IUC.

Acknowledgement

This study was supported by the Project IGA no. 9678-4.

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